In the "baby machine" culture system, cells are immobilized on a surface such that newborn cells are shed continuously from the culture. When an immobilized cell divides, one of the new progeny cells remains attached and the other is released. This culture system has been used to decipher many of the cell cycle/DNA replication-segregation/cell division properties of E. coli B/r. Its usefulness has been limited, however, by the number of newborn cells released and the inability to use the many E. coli K12 derivatives. Recognizing that both problems required solutions to make real headway in solving regulatory aspects of the cell cycle, the baby machine has been modified to function with most microorganism, and the cell yield has been increased 5 to 20-fold. Accordingly, a variety of K12 derivatives will be used to determine molecular mechanisms involved in the control of chromosome replication/segregation and cell division. Among the projects to be undertaken are: 1) Continued modifications of the baby machine, primarily to develop covalent attachment of the cells, in order to develop its full potential for cell cycle/cell aging studies with a variety of species; 2) Analyze cell cycle parameters of K12 derivatives and identify the interactions among the components of the circuit controlling the timing of initiation of chromosome replication; 3) Test theories on control of the timing and toporegulation of cell division with K12 derivatives; 4) Determine the replication patterns of low copy F and PI plasmids in the cell. cycle; 5) Identify the determinants of nonrandom chromosome segregation; 6) Examine OriC-envelope interactions in relation to initiation of replication and segregation; 7) Determine the involvement of superhelicity in minichromosome stability and partitioning; 8) Assay cell cycle-specific transcription of genes. involved In initiation of chromosome replication; 9) Identify the cell cycle-dependent appearance of modulators of chromosome replication in an in vitro minichromosome replication system. The ability to perform physiologically sound cell cycle studies using strain K12 with improved yields should help resolve many unanswered questions on the control and coordination between chromosome replication/segregation and cell division in E. coli.